The ability of the HIV-associated fungal pathogen Histoplasma capsulatum (Hc) to cause severe invasive disease is attributed to the fungus' ability to efficiently subvert host responses. For example, the capacity of the fungus to impair phagosome maturation and the presence of melanin in the fungal cell wall contribute to intracellular survival through avoidance of host surveillance as well as blocking the effects of host effector responses. We have shown that Hc produces extracellular vesicles containing biologically active molecules. However, many questions on disease pathogenesis arise from this process of vesiculogenesis. In the first Aim, we will explore how protective and non-protective antibodies (Abs) regulate the production and release of extracellular vesicles from Hc. We are pursuing this work based on our findings that protective Abs to Hc alter gene expression and that cross-linking by Abs changes the outcome of interactions with host effector cells. Using a pair of protective and non-protective monoclonal antibodies (mAbs) that bind to the same region on heat shock protein 60 expressed on the Hc cell surface, we will determine (Aim 1) the mAbs' capacity to alter the formation and secretion of these bilayered vesicles as well as (Aim 2) their impact on the protein and lipid cargo within the vesicles. We are pursuing this work based on our observations that the vesicles can alter the behavior of host cells and that the cargo within the vesicles includes compounds that are immunomodulatory. The results of the studies in this R21 will provide new insights into the basic biology of Hc, elucidate new activities of Abs, and serve as a potential foundation for new therapeutic approaches to combat this important fungus as well as other eukaryotic pathogens.